The invention relates to a method for replicating and analyzing one or a plurality of different target sequences in nucleic acid sequences and apparatus including an array to be selectively heated for replicating and analyzing a plurality of target sequences in nucleic acid samples.
It is known that when analyzing nucleic acid sequences, the latter must almost always be multiplied because frequently too few copies of the nucleic acid sequences sought are present in the initial sample. In addition, selective reproduction of the sequences to be analyzed facilitates their detection in the presence of a great excess of other nucleic acid sequences.
Replication is generally performed using the polymerase chain reaction (PCR). For this, the complete sample solution is alternately heated and cooled cyclically in order to permit the steps of denaturing the double strand (approx. 95° C.), hybridizing the primer (e.g. 50° C.), and extending the primer (e.g. 75° C.) to run repeatedly one after the other.
So-called thermocyclers are commercially available for this purpose; these are devices in which the reaction is performed in small, typically 500-μL, reaction vessels. The reaction vessels can be combined to create palettes to enable parallel processing of a plurality of samples. Miniaturized analysis systems in which the PCR is performed are also described.
Kopp et al. describe a hydrostatically operated flow system with three different heated zones. A flow channel is configured such that it alternately passes through the three set temperature areas so that in this manner the temperature cycle is created for the PCR (M.U. Kopp, A.D. de Mello, A. Manz: “Chemical Amplification: Continuous-Flow PCR on a Chip”, Science 280 (1198) 1046).
Z. Chen et al. provide a similar system, in this case the movement of the liquid being initiated by the thermosyphon principle (Z. Chen, S. Qian, W. R. Abrams, D. Malamud, H. H. Bau, Anal. Chem. 76 (2004) 3707).
Liu et al. describe a printed circuit board-based integrated analysis system that combines both sample preparation and DNA multiplication and microarray detection (R. H. Liu, J. Yang, R. Lenigk, J. Bonanno, P. Grodzinski: “Self-Contained, Fully Integrated Biochip for Sample Preparation, Polymerase Chain Reaction Amplification, and DNA Microarray Detection”, Analytical Chemistry 76 (2004) 1824).
In the strand displacement amplification (SDA) method, cyclical heating and cooling of the sample are not needed. Special nick enzymes and primers at a constant temperature ensure that the double strands are separated by newly formed complementary strands and the old complementary strand is displaced (SDA with thermophilic enzymes, U.S. Pat. No. 5,648,211; SDA combined with bioelectronic chips U.S. Pat. No. 6,238,868 B1).
Fluorimetric analysis of the replicated target strands can be performed simultaneous to the PCR. In this real-time PCR, the nearly complete PCR process is not required, but rather it is possible to draw conclusions about the content of nucleic acids with the target sequence in the starting material just from the temporal course of the analysis signal.
The analysis of nucleic acids can also be performed on selectively heatable electro-chemical analysis arrays, such as are described in U.S. Pat. No. 6,255,677 B1 and DE application 10 2004 017750.
Also known are analysis systems in which both replication and also analysis occur on one array. U.S. Pat. No. 6,326,173 B1 and Nat. Biotechnol. 18 (2000) 199 describe e.g. electrically induced hybridization of the target with the primer. The primers can be dissolved homogeneously or immobilized. Replication occurs using SDA. Optical methods are used for the analysis.
Primarily the great degree of complexity in terms of time and equipment are disadvantageous in most of the known methods, as is the complicated technical operation. In addition, the replication step generally occurs separate from the analysis step in terms of both time and equipment.
For this reason widespread use of conventional DNA sequence analysis technology for instance in medicine has not been possible in the past.
In all of the previous PCR variants it has been a hindrance that all of the components for the PCR analysis solution must go through the entire temperature cycle. Therefore only certain thermally stable polymerases can be used for the primary extension, which increases costs and limits versatility. One alternative is to re-add the polymerase after each cycle, which is very inconvenient.
Otherwise the analysis is generally performed using optical methods that increase the complexity of the equipment, or using separating methods (electrophoresis) that do not permit parallel determination of a large number of different target sequences, or only permit it with difficulty.
However, a rapid and simple method for parallel determination of many target sequences (even in trace amounts) is desirable. A miniaturized analysis system with integrated PCR or SDA that permits determination of a plurality of target sequences would be very advantageous. For instance, individual therapy with targeted and sparing use of medications would be possible. Costs and side-effects could be reduced, and the success of therapy could be improved. Development of medications would also profit greatly from such a novel method because frequently gene expression must be monitored for testing new drugs.